Summary of Work: Rare replication errors are corrected by the cells spellchecking machinery, post-replication DNA mismatch repair (MMR). The goal of this project is to understand the biochemistry and genetics of MMR in normal and mutant eukaryotic cells. This year we established a system to study the funtion of human mismatch repair proteins expressed in yeast. We performed functional studies of MLH1 demonstrating that overexpression in wild-type cells yields a dominant mutator effect, that 6 of 6 HNPCC-associated missense mutations completely inactivate mismatch repair and all map to the protein structure at locations consistent with loss of ATP binding and/or hydrolysis, that MLH1 (+/-) diploid cells have an elevated mutation rate that results from loss of heterozygosity (LOH), and that LOH can be promoted by a DNA damaging agent (bleomycin). We identified the first MSH2 missense mutations that exhibit a dominant mutator effect when expressed from the natural MSH2 gene promoter located on the chromosome, including one missense mutation found in a colon cancer patient. We further demonstrated that MSH2 (+/-) diploid cells have an elevated mutation rate that can be synergistically elevated by a mutation in a replicative DNA polymerase that partially inactivates proofreading of replication errors. These studies are important for understanding the genetics and biochemistry of the mismatch repair system, the multiple functions of the numerous mismatch repair genes, the molecular genetic basis for the initiating events in cancer and its subsequent treatment and the risk posed to individuals in the population by exposure to DNA damaging agents. - Cancer, DNA Damage, Methylation, Microsattelite Instability, Replication Fidelity, Mismatch Repair